Provided with each kit are four 96-well plates that come individually packaged in foil pouches. The plates contain a lyophilized mixture of Xfect Transfection Reagent and Lenti-X packaging plasmids that has been precisely aliquoted into each well. The plates are sealed with a pierceable foil that allows for processing on automated liquid handling platforms.
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Streamlined production of high-titer lentivirus in a 96-well format
Lenti-X Packaging Single Shots (96-well, VSV-G)
Production of lentiviral vectors in a 96-well format is a common starting point for a variety of high-throughput applications, including arrayed CRISPR or shRNA-based screening and vector optimization workflows. However, obtaining sufficient viral titers in a high-throughput context can prove challenging, as production processes typically involve optimizing proportions of the respective packaging reagents and distributing them in minute quantities. To help researchers overcome these obstacles and streamline their process development efforts, we have adapted our popular Lenti-X packaging system to enable consistent high-titer production of VSV-G-pseudotyped lentiviral particles in 96-well plates. Lenti-X Packaging Single Shots (96-well, VSV-G) consist of a lyophilized mixture of Xfect Transfection Reagent and optimized ratios of Lenti-X packaging plasmids, pre-aliquoted in a 96-well format, providing researchers with the following benefits:
- High-throughput lentivirus production made fast and simple—just add transfer vector plasmid to each well, mix, and apply to packaging cells to produce lentivirus; avoid spending time formulating and aliquoting transfection mixes on your own
- Consistently high viral titers—our system typically yields titers of 1 x 107–1 x 108 IFU/ml, and usable titers are obtainable even under suboptimal conditions (e.g., lower-than-anticipated inputs of transfer vector plasmid)
- High well-to-well and lot-to-lot uniformity—functional testing demonstrated a well-to-well coefficient of variation (CV) of less than 15% for successive lots of Lenti-X Packaging Single Shots (96-well, VSV-G), so users can expect consistent performance from each and every plate
What is it?
How does it work?
Lenti-X Packaging Single Shots (96-well, VSV-G) provide a streamlined method for high-throughput lentivirus production and are compatible with all commonly available lentiviral transfer vector backbones. Users simply add 10 µl of plasmid encoding their transfer vector of choice to each well and mix to solubilize the pre-aliquoted transfection mixes. The resulting nanoparticle complexes are then used to transfect packaging cells (e.g., Lenti-X 293T cells) in a separate plate. After 48–72 hours, high-titer lentivirus can be harvested from the supernatant in each well and used to transduce target cells in a separate plate. This workflow is summarized by the following figure:
What are typical titers?
Lenti-X Packaging Single Shots (96-well, VSV-G) employ Takara Bio's proprietary fourth-generation lentiviral packaging system, which has been extensively optimized to maximize resulting titers. In a 96-well context, this system typically yields titers of 1 x 107–1 x 108 IFU/ml, as demonstrated by the data below.
How consistent are results?
To effectively support high-throughput workflows and avoid the introduction of experimental artifacts, a product must provide consistent performance from well to well and plate to plate. To ensure the reliability of Lenti-X Packaging Single Shots (96-well, VSV-G) for high-throughput lentivirus production, well-to-well variation in transfection efficiencies was analyzed using plates from two different production lots.
How robust is the workflow?
Given the reality that quantitation and adjustment of transfer vector plasmid concentrations may not be practical in the context of a high-throughput workflow, it is helpful to have a production system that still yields usable titers despite variations in this regard. With this in mind, viral titer production with Lenti-X Packaging Single Shots (96-well, VSV-G) was evaluated across a range of transfer vector plasmid input amounts.
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